Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA-RNA recombination

ABSTRACT RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5′-3′ exoribonuclease, whose absence led to an ∼10- to 50-fold enhancement of RNA recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5′-truncated viral RNAs in xrn1Δ mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1Δ mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5′-truncated viral RNAs were expressed in the parental and xrn1Δ mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5′-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA recombination by rapidly removing the 5′-truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5′-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus recombination and evolution.

Download Full-text

CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases

RNA ◽

10.1017/s1355838200992410 ◽

2000 ◽

Vol 6 (5) ◽

pp. 698-707 ◽

Cited By ~ 31

Author(s):

SHIGEO YOSHINARI ◽

PETER D. NAGY ◽

ANNE E. SIMON ◽

THEO W. DREHER

Keyword(s):

In Vitro Transcription ◽

Rna Polymerases ◽

Viral Rna ◽

Promoter Elements

Download Full-text

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

Journal of Virology ◽

10.1128/jvi.02620-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2750-2763 ◽

Cited By ~ 11

Author(s):

K. Reddisiva Prasanth ◽

Daniel Barajas ◽

Peter D. Nagy

Keyword(s):

Viral Replication ◽

Genetic Recombination ◽

Rna Virus ◽

Viral Rna ◽

Rna Recombination ◽

Viral Recombination ◽

Antiviral Responses ◽

New Hosts

ABSTRACTRNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination inSaccharomyces cerevisiaeand plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast orin vitrobased on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92polreplication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed inrpn11mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs.IMPORTANCERNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, andin vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination.

Download Full-text

Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

Chemical Communications ◽

10.1039/c4cc08862j ◽

2015 ◽

Vol 51 (37) ◽

pp. 7887-7890 ◽

Cited By ~ 17

Author(s):

Hideto Maruyama ◽

Kazuhiro Furukawa ◽

Hiroyuki Kamiya ◽

Noriaki Minakawa ◽

Akira Matsuda

Keyword(s):

Nucleic Acids ◽

Mammalian Cells ◽

Genetic Material ◽

Rna Polymerases ◽

Chemically Modified ◽

Biological Studies ◽

Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

Download Full-text

Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

International Journal of Molecular Sciences ◽

10.3390/ijms22010058 ◽

2020 ◽

Vol 22 (1) ◽

pp. 58

Author(s):

Thomas Gremminger ◽

Zhenwei Song ◽

Juan Ji ◽

Avery Foster ◽

Kexin Weng ◽

...

Keyword(s):

Reverse Transcription ◽

Potential Interaction ◽

Viral Rna ◽

Primer Binding Site ◽

Rna Integrity ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Extended Interaction ◽

Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Download Full-text

Unusual requirements for optimum translation of polio viral RNA in vitro.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)32351-2 ◽

1983 ◽

Vol 258 (11) ◽

pp. 7195-7199 ◽

Cited By ~ 1

Author(s):

S Daniels-McQueen ◽

B M Detjen ◽

J A Grifo ◽

W C Merrick ◽

R E Thach

Keyword(s):

Viral Rna

Download Full-text

Studies of two temperature-sensitive mutants of Mengo virus

Canadian Journal of Biochemistry ◽

10.1139/o79-110 ◽

1979 ◽

Vol 57 (6) ◽

pp. 902-913 ◽

Cited By ~ 1

Author(s):

Patrick W. K. Lee ◽

John S. Colter

Keyword(s):

Rna Synthesis ◽

Viral Rna ◽

Temperature Sensitive ◽

Supporting Evidence ◽

Structural Polypeptide ◽

Infected Cells ◽

Mengo Virus ◽

In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

Download Full-text

RNAP subunits F/E (RPB4/7) are stably associated with archaeal RNA polymerase: using fluorescence anisotropy to monitor RNAP assembly in vitro

Biochemical Journal ◽

10.1042/bj20090782 ◽

2009 ◽

Vol 421 (3) ◽

pp. 339-343 ◽

Cited By ~ 20

Author(s):

Dina Grohmann ◽

Angela Hirtreiter ◽

Finn Werner

Keyword(s):

Rna Polymerase ◽

Molecular Interactions ◽

Fluorescence Anisotropy ◽

Rna Polymerases ◽

Dynamic Exchange ◽

Transcription Cycle

Archaeal and eukaryotic RNAPs (DNA-dependent RNA polymerases) are complex multi-subunit enzymes. Two of the subunits, F and E, which together form the F/E complex, have been hypothesized to associate with RNAP in a reversible manner during the transcription cycle. We have characterized the molecular interactions between the F/E complex and the RNAP core. F/E binds to RNAP with submicromolar affinity and is not in a dynamic exchange with unbound F/E.

Download Full-text